1. Field of the Invention
The present invention is broadly concerned with methods for purifying synthetic peptides in which reversible alterations of the physicochemical properties of the peptides are exploited. More particularly, in preferred embodiments, a polymer is added to a desired peptide either by condensation with preformed polymer or by in situ polymerization such that a cleavable linker is interposed between the polymer moiety and the peptide moiety of the resultant polymer-peptide adduct; the adduct is then easily purified from undesired, nonadducted peptides since the adduct and the nonadducted peptides have different solubility characteristics; subsequently, the adduct may be cleaved at the linker and the desired peptide purified from the polymer.
2. Description of the Prior Art
The demand for synthetic peptides in many research fields has increased in recent years, particularly since the advent of solid-phase peptide synthesis. For example, in immunochemistry, peptides as epitopes of proteins are important for antibody production. Also, peptides are often used as ligands in affinity chromatography.
In solid-phase peptide synthesis, amino acids are coupled in stepwise fashion to a peptide attached to an insoluble support contained within a reaction vessel. The insoluble support is composed of a resin which is typically polystyrene. Each successive coupling of an amino acid is carried out by passing the amino acid through the reaction vessel. The amino acid is "protected," i.e., it has a stable but reversible blocking group attached to it which prevents polymerization of that amino acid. Suitable reversible blocking groups include benzyloxycarbonyl, t-butyloxycarbonyl (BOC), and 9-fluorenylmethoxycarbonyl (Fmoc) groups. After the amino acid is coupled to the peptide, the reversible blocking group is removed to allow addition of another amino acid to the peptide.
Amino acid coupling reactions are highly efficient; more than 99% of the peptides have an amino acid added to them during each coupling cycle. However, a small percentage of the peptides fail to receive an amino acid during each coupling cycle; these peptides, referred to as "failed sequences," represent a serious problem in the synthesis of peptides. A 1% failure rate per coupling cycle gives a significant amount of failed sequences at the end of a synthesis having multiple cycles.
For example, a failure rate of 1% per cycle in the syntheses of a 30-residue peptide (29 cycles) and a 50-residue peptide (49 cycles) generates greater than 22% and 39% by weight of failed sequences, respectively, in the final mixtures. Furthermore, a variety of failed sequences different from each other are generated which may be deficient in as little as one residue as compared with the target peptide. It is very difficult, if not impossible, to separate the target peptide from the failed sequences using normal separation techniques since the target peptide and the failed sequences may have nearly identical physicochemical properties. Typically, specifications for peptides call for a purity ranging from 95 to 98%. This level of purity can be attained for peptides having less than 30 residues using high performance liquid chromatography (HPLC) as the purification method. However, this level of purity cannot be attained for larger peptides using HPLC or other state-of-the-art purification techniques.
In the past, this separation problem has been alleviated somewhat by passing a "capping" reagent through the reaction vessel in order to "cap" failed sequences. The presence of the reversible blocking group on a coupled amino acid prevents capping of the elongating target peptide. This cap prevents failed sequences from participating in subsequent coupling cycles. Thus, the sequences that fail early in the peptide synthesis are substantially shorter than the target peptide and therefore have different physicochemical properties that can be exploited in separation techniques. Typical capping reagents include acetic anhydride and 4-methoxybenzoic acid.
Despite the use of capping reagents, separation of the target peptide from failed sequences remains a serious problem. Affinity chromatography methods have been proposed wherein a target peptide is biotinylated while failed sequences are not Journal of Chromatography, 638:21-27 (1993); Tetrahedron Letters, 36:9097-9100 (1995)!. The biotinylated peptide and the failed sequences are then cleaved from the support and passed through an expensive avidin-agarose column to adsorb the biotinylated peptide. The adsorbed biotinylated peptide is then eluted from the column and treated with base to release the biotin moiety and yield the target peptide. These methods have not gained wide acceptance because the presence of the biotin moiety does little to alter the physicochemical properties of the target peptide, and the avidin-agarose column is very expensive and has a limited life-time.
There is accordingly an unsatisfied need in the art for a method for the synthesis and purification of peptides which is simple, uses relatively inexpensive reagents, and provides the desired peptide in good yield and at a high level of purity.